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Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

Gomez GA, Veldman MB, Zhao Y, Burgess S, Lin S - PLoS ONE (2009)

Bottom Line: Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel.Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development.The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

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Morpholino knockdown of etsrp reduces expression of genes identified by etsrp overexpression microarray analysis.Embryos were injected with a combination of two etsrp targeting morpholinos (4 ng each) and gene expression was examined by in situ hybridization at 24–26 hours post fertilization. Representative control embryos are on the left of each panel and etsrp morpholino (MO) injected embryos are on the right. (A) krml2 is reduced in primitive myeloid cells, posterior cardinal vein, and caudal hematopoietic tail region by etsrp knockdown. (B) lrrp33 expression is absent in myeloid cells and reduced in the caudal hematopoietic tail region. (C) hapln1b expression is lost in the posterior cardinal vein when etsrp is knocked down. (D–N) Etsrp knockdown causes a significant decrease of gene expression, most pronounced in the trunk (unlabeled arrows). (D) rasgfp3; (E) tem8; (F) arhgap29; (G) fgd5; (H) yrk; (I) similar to hemicentin; (J) sh3gl3; (K) similar to costimulatory protein; (L) ldb2; (M) est:AI7201944; and (N) est:AW019729. Note that non-hematovascular tissues such as somites in (A) and hypochord in (C) are not affected by etsrp knockdown. Abbreviations: pm, primitive myeloid; pcv, posterior cardinal vein; cht, caudal hematopoietic tail region; ss, somites; and hc, hypochord. Scale bar: 250 µm.
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pone-0004994-g005: Morpholino knockdown of etsrp reduces expression of genes identified by etsrp overexpression microarray analysis.Embryos were injected with a combination of two etsrp targeting morpholinos (4 ng each) and gene expression was examined by in situ hybridization at 24–26 hours post fertilization. Representative control embryos are on the left of each panel and etsrp morpholino (MO) injected embryos are on the right. (A) krml2 is reduced in primitive myeloid cells, posterior cardinal vein, and caudal hematopoietic tail region by etsrp knockdown. (B) lrrp33 expression is absent in myeloid cells and reduced in the caudal hematopoietic tail region. (C) hapln1b expression is lost in the posterior cardinal vein when etsrp is knocked down. (D–N) Etsrp knockdown causes a significant decrease of gene expression, most pronounced in the trunk (unlabeled arrows). (D) rasgfp3; (E) tem8; (F) arhgap29; (G) fgd5; (H) yrk; (I) similar to hemicentin; (J) sh3gl3; (K) similar to costimulatory protein; (L) ldb2; (M) est:AI7201944; and (N) est:AW019729. Note that non-hematovascular tissues such as somites in (A) and hypochord in (C) are not affected by etsrp knockdown. Abbreviations: pm, primitive myeloid; pcv, posterior cardinal vein; cht, caudal hematopoietic tail region; ss, somites; and hc, hypochord. Scale bar: 250 µm.

Mentions: The dependence of the new blood and vascular specific genes on etsrp was then examined by comparing their expression in etsrp morphants to uninjected control embryos (Figure 5). In etsrp morphants, the expression of krml2 (Figure 5A) and lrrp33 (Figure 5B) was abolished in myeloid cells, while the remaining 12 vascular genes were also significantly reduced in expression, most noticeably in the trunk region containing the dorsal aorta, posterior cardinal vein, and intersomitic vessels (Figure 5, C–N). Note that gene expression in the cranial vasculature was less affected in etsrp morphants but still reduced. Gene expression in tissues outside of blood and vessels such as somites for krml2 (Figure 5A), neurons for rasgrp3 (Figure 5D) and ldb2 (Figure 5L), and tailbud for tem8 (Figure 5E) were not affected by etsrp knockdown. This demonstrates that etsrp is not only sufficient but also required for normal expression in primitive myeloid and/or vascular cells in the 14 genes identified here.


Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

Gomez GA, Veldman MB, Zhao Y, Burgess S, Lin S - PLoS ONE (2009)

Morpholino knockdown of etsrp reduces expression of genes identified by etsrp overexpression microarray analysis.Embryos were injected with a combination of two etsrp targeting morpholinos (4 ng each) and gene expression was examined by in situ hybridization at 24–26 hours post fertilization. Representative control embryos are on the left of each panel and etsrp morpholino (MO) injected embryos are on the right. (A) krml2 is reduced in primitive myeloid cells, posterior cardinal vein, and caudal hematopoietic tail region by etsrp knockdown. (B) lrrp33 expression is absent in myeloid cells and reduced in the caudal hematopoietic tail region. (C) hapln1b expression is lost in the posterior cardinal vein when etsrp is knocked down. (D–N) Etsrp knockdown causes a significant decrease of gene expression, most pronounced in the trunk (unlabeled arrows). (D) rasgfp3; (E) tem8; (F) arhgap29; (G) fgd5; (H) yrk; (I) similar to hemicentin; (J) sh3gl3; (K) similar to costimulatory protein; (L) ldb2; (M) est:AI7201944; and (N) est:AW019729. Note that non-hematovascular tissues such as somites in (A) and hypochord in (C) are not affected by etsrp knockdown. Abbreviations: pm, primitive myeloid; pcv, posterior cardinal vein; cht, caudal hematopoietic tail region; ss, somites; and hc, hypochord. Scale bar: 250 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654924&req=5

pone-0004994-g005: Morpholino knockdown of etsrp reduces expression of genes identified by etsrp overexpression microarray analysis.Embryos were injected with a combination of two etsrp targeting morpholinos (4 ng each) and gene expression was examined by in situ hybridization at 24–26 hours post fertilization. Representative control embryos are on the left of each panel and etsrp morpholino (MO) injected embryos are on the right. (A) krml2 is reduced in primitive myeloid cells, posterior cardinal vein, and caudal hematopoietic tail region by etsrp knockdown. (B) lrrp33 expression is absent in myeloid cells and reduced in the caudal hematopoietic tail region. (C) hapln1b expression is lost in the posterior cardinal vein when etsrp is knocked down. (D–N) Etsrp knockdown causes a significant decrease of gene expression, most pronounced in the trunk (unlabeled arrows). (D) rasgfp3; (E) tem8; (F) arhgap29; (G) fgd5; (H) yrk; (I) similar to hemicentin; (J) sh3gl3; (K) similar to costimulatory protein; (L) ldb2; (M) est:AI7201944; and (N) est:AW019729. Note that non-hematovascular tissues such as somites in (A) and hypochord in (C) are not affected by etsrp knockdown. Abbreviations: pm, primitive myeloid; pcv, posterior cardinal vein; cht, caudal hematopoietic tail region; ss, somites; and hc, hypochord. Scale bar: 250 µm.
Mentions: The dependence of the new blood and vascular specific genes on etsrp was then examined by comparing their expression in etsrp morphants to uninjected control embryos (Figure 5). In etsrp morphants, the expression of krml2 (Figure 5A) and lrrp33 (Figure 5B) was abolished in myeloid cells, while the remaining 12 vascular genes were also significantly reduced in expression, most noticeably in the trunk region containing the dorsal aorta, posterior cardinal vein, and intersomitic vessels (Figure 5, C–N). Note that gene expression in the cranial vasculature was less affected in etsrp morphants but still reduced. Gene expression in tissues outside of blood and vessels such as somites for krml2 (Figure 5A), neurons for rasgrp3 (Figure 5D) and ldb2 (Figure 5L), and tailbud for tem8 (Figure 5E) were not affected by etsrp knockdown. This demonstrates that etsrp is not only sufficient but also required for normal expression in primitive myeloid and/or vascular cells in the 14 genes identified here.

Bottom Line: Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel.Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development.The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

Show MeSH
Related in: MedlinePlus